The invention relates to the use of fluorescent proteins, particularly green fluorescent protein (GFP), in fusion constructs with random and defined peptides and peptide libraries, to increase the cellular expression levels, decrease the cellular catabolism, increase the conformational stability relative to linear peptides, and to increase the steady state concentrations of the random peptides and random peptide library members expressed in cells for the purpose of detecting the presence of the peptides and screening random peptide libraries. N-terminal, C-terminal, dual N- and C-terminal and one or more internal fusions are all contemplated. Novel fusions utilizing self-binding peptides to create a conformationally stabilized fusion domain are also contemplated.
The field of biomolecule screening for biologically and therapeutically relevant compounds is rapidly growing. Relevant biomolecules that have been the focus of such screening include chemical libraries, nucleic acid libraries and peptide libraries, in search of molecules that either inhibit or augment the biological activity of identified target molecules. With particular regard to peptide libraries, the isolation of peptide inhibitors of targets and the identification of formal binding partners of targets has been a key focus. However, one particular problem with peptide libraries is the difficulty assessing whether any particular peptide has been expressed, and at what level, prior to determining whether the peptide has a biological effect.
Green fluorescent protein (GFP) is a 238 amino acid protein. The crystal structure of the protein and of several point mutants has been solved (Ormo et al., Science 273, 1392-5, 1996; Yang et al., Nature Biotechnol. 14, 1246-51, 1996). The fluorophore, consisting of a modified tripeptide, is buried inside a relatively rigid beta-can structure, where it is almost completely protected from solvent access. The fluorescence of this protein is sensitive to a number of point mutations (Phillips, G. N., Curr. Opin. Struct. Biol. 7, 821-27, 1997). The fluorescence appears to be a sensitive indication of the preservation of the native structure of the protein, since any disruption of the structure allowing solvent access to the fluorophoric tripeptide will quench the fluorescence.
Abedi et al (Nucleic Acids Res. 26, 623-30, 1998) have inserted peptides between residues contained in several GFP loops. Inserts of the short sequence LEEFGS (SEQ ID NO:1) between adjacent residues at 10 internal insertion sites were tried. Of these, inserts at three sites, between residues 157-158, 172-173 and 194-195 gave fluorescence of at least 1% of that of wild type GFP. Only inserts between residues 157-158 and 172-173 had fluorescence of at least 10% of wild type GFP. When -SAG-random 20 mer-GAS- peptide sequences (SEQ ID NO:2) were inserted at different sites internal to GFP, only two sites gave mean fluorescence intensities of 2% or more of the GFP-random peptide sequences 10-fold above background fluorescence. These sites were insertions between residues 157-158 and 172-173.
It is an object of the invention to provide compositions of fusion constructs of peptides with fluorescent proteins such as GFP, and methods of using such constructs in screening of peptide libraries.
In accordance with the objects outlined above, the present invention provides fusion proteins comprising a random peptide fused to green fluorescent protein (GFP). Preferred embodiments utilize fusions to the N- and C-termini of GFP comprising presentation structures capable of presenting the peptide in a conformationally restricted form. Further preferred embodiments fuse the random peptide to an internal position of GFP, including the loops comprising amino acids 130 to 135, amino acids 154 to 159, amino acids 172 to 175, amino acids 188 to 193, and amino acids 208 to 216.
In a further aspect, the invention provides fusion nucleic acids encoding the fusion proteins.
In an additional aspect, the present invention provides libraries of: a) fusion proteins; b) fusion nucleic acids; c) expression vectors comprising the fusion nucleic acids; and d) host cells comprising the fusion nucleic acids.
In a further aspect, the invention provides methods of screening for bioactive peptides confering a particular phenotype. The methods comprise providing cells containing a fusion nucleic acid comprising nucleic acid encoding a fusion protein comprising GFP and a random peptide as above. The cells are subjected to conditions wherein the fusion protein is expressed. The cells are then assayed for the phenotype.